Study On The Biological Effect Of Tanshinone ?A On Human Retinal Endothelial Cells Cultured With High Glucose In Vitro

Objectives: Diabetic retinopathy(DR)is the most common and serious complication of diabetes mellitus.In this study,human retinal endothelial cells(HRECs)were used to study the effects of high glucose environment on the proliferation,migration and angiogenesis of human retinal endothelial cells in vitro.Meanwhile,the biological effect of Tanshione IIA(TSA)was investigated by observing changes of proliferation of HRECs in the environment of normal and high glucose respectively.Additionally,the biological effect of TSA on migration and angiogenesis of HRECs was studied in the environment of high glucose.To explore potential mechanisms of the prevention and treatment of diabetic retinopathy by TSA,the effects of TSA on the transcription and expression of VEGF and ICAM-1 in HRECs were further evaluated.Methods: 1.The CCK8 method was used to observe the proliferation ability of HRECs which were cultured with normal glucose(NG)medium and high glucose(HG)medium respectively under different concentrations of tanshinone IIA(TSA).The concentration of TSA was 10,20 and 30(?g/ml),and the CCR8 checking points were 24 h,48 h and 72 h respectively.2.The changes of migration ability of HRECs in high glucose environment under different tanshinone IIA(TSA)concentrations were measured by wound healing and transwell experiments.The wound healing was recorded by inverted fluorescence microscopy at 0 h,24 h,48 h.Transwell experiments were performed to record the number of migrating cells in all groups after 24 h.3.The effect of TSA on tube formation of HRECs was detected under different TSA concentrations in high glucose environment.4.The levels of VEGF and ICAM-1 m RNA and protein of HRECs in different TSA concentrations were analyzed by q RT-PCR,Western blot and Immunofluorescence technique.5.All data were statistically analyzed using SPSS 16.0 software.The quantitative data were expressed as mean ± standard deviation(x ± s),using the independent sample t test for two sets of data and one-way analysis of variance for multi group data.For the consecutive data,the method of variance analysis of repeated measurement was used.LSD methods were used for further comparison of two sets of data.P <0.05 was considered statistically significant.Results: 1.In the CCK8 experiment,the high glucose environment promotes the proliferation of HRECs;the proliferation ability of HRECs was not affected by Tanshinone IIA(TSA)at normal glucose concentration.In high glucose environment,the proliferative ability of HRECs was suppressed by TSA.TSA inhibit the proliferation of HRECs in a time-dependent and dose-dependent manner.At 72 h,HG-30 group showed the most powerful effect of inhibition.2.The results of wound healing study were consistent with the transwell experiment.Compared with the normal glucose environment,the high glucose could promote the migration of HRECs.In the high glucose environment,TSA inhibited cell migration in a dose-dependent manner.HG-30 group showed the most evident inhibition effect.3.In vitro,the number and size of tube formation in high glucose environment were higher than those in normal glucose environment.The tube formation ability of HRECs in high glucose environment was inhibited by TSA in a dose-dependent manner.In HG-30 group,the inhibition effect was most obvious.4.The results of q RT-PCR,Western blot and Immunofluorescence showed that the m RNA and protein expression levels of VEGF and ICAM-1 were increased in high glucose induced HRECs compared with normal glucose concentration.However,in the environment of high glucose,TSA significantly downregulated the m RNA and protein expression levels of VEGF in a dose-dependent manner.And TSA additionally significantly decreased the protein expression level of ICAM-1 in a dose-dependent manner.The effect of HG-30 group is the most obvious.Conclusions: 1.Compared with normal glucose environment,the proliferation,migration and tube formation ability of HRECs were promoted under high glucose conditions.2.TSA has a certain inhibitory effect on the proliferation ability of HRECs cultured with high glucose in a time-dependent and dose-dependent manner.And TSA has a certain inhibitory effect on the migration and tube formation ability of HRECs cultured with high glucose in a dose-dependent manner.Importanly,TSA manifested no inhibition effect on HRECs proliferation in the circumstance of normal glucose.3.The m RNA and protein expression levels of VEGF and ICAM-1 was increased in high glucose induced HRECs.TSA inhibited the m RNA and protein expression levels of VEGF and ICAM-1 in high glucose induced HRECs,and the effect was also in a dose-dependent manner.